title: Purification and some properties of a thermostable DNA polymerase from a Thermotoga species.
abstract: A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.
authors.affiliation: Department Biological Sciences, University of Waikato, Hamilton, New Zealand.
authors.forename: H D
authors.initials: HD
authors.lastname: Simpson
authors.forename: T
authors.initials: T
authors.lastname: Coolbear
authors.forename: M
authors.initials: M
authors.lastname: Vermue
authors.forename: R M
authors.initials: RM
authors.lastname: Daniel
mesh_terms: D000693:Anaerobiosis; D003080:Cold Temperature; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight
chemical_list: D004259:DNA-Directed DNA Polymerase